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activator ahrC from Bacillus subtilis
J. Mot. Biol. (1990) 213,227-228
Crystallization of the Arginine-dependent Repressor/Activator AhrC from Bacillus subtilis C. W. G. Boys 1, L. G. Czaplewski 2, S. E. V. Phillips 1, S. Baumberg 2 and P. G. Stockley 2 I Astbury Department of Biophysics 2Department of Genetics University of Leeds, Leeds L S 2 9 J T , U.K. (Received 18 J a n u a r y 1990; accepted 24 J a n u a r y 1990) The arginine-dependent repressor/activator AhrC from Bacillus subtilis has been crystallized in space group C2221, with unit cell dimensions a=229"8 A, b=72-8 A, c= 137"7 A and one aporepressor hexamer per asymmetric unit. Preliminary X-ray photographs show measurable intensities beyond 3"0 A.
dependent r~pressor/activator AhrC from BaciUis subtilis in a form that diffracts to high resolution and may allow a full three-dimensional structure determination. AhrC is the product of the ahrC locus and has been shown to repress the transcription of the arginine biosynthetic enzymes in an arginine-dependent manner, as well as activating the transcription of enzymes involved in arginine catabolism (Mountain & Baumberg, 1980; North et al., 1989). AhrC protein is a hexamer of subunit molecular mass 16"9 kDa (Czaplewski et al., unpublished results). The sequence displays 27 ~ identity with the E. coli homologue ArgR, which has also been shown to be a hexamer (Lim et al., 1987). In addition to regulating arginine biosynthesis in E. coli, ArgR is involved in a site-specific recombination reaction (Stifling et al., 1988). AhrC will complement argR- E. coli strains, although it is not as active as ArgR {Stifling et al., 1988; Smith et al., 1989). No recombination function for the protein in B. subtilis has been discovered, although the gene does lie adjacent to at least one other gene having strong homology with enzymes involved in recombination/repair in E. coli (North, 1989; Van H o y & Hoeh, unpublished results). The AhrC sequence does not contain features characteristic of a helix-turnhelix motif or significant homology to other known repressor proteins, and thus may be representative of a different class of DNA-binding proteins from those studied structurally. The elucidation of its three-dimensional structure should add to our understanding of DNA-protein interactions. Cloned ahrC was overexpressed in E. coli and the protein purified by differential precipitation followed by chromatography on S-Sepharose, which
There are a growing number of known threedimensional structures of bacterial regulatory proteins (Anderson et al., 1981; Pabo & Lewis, 1982), including several complexed with DNA oligonucleotides (Otwinowski et al., 1988; Jordan & Pabo, 1988; Aggarwal et al., 1988). To date, all these structures are of small dimeric molecules isolated from Escherichia coli or its lambda phages and, until very recently, all known structures belonged to the helixturn-helix family of DNA-binding proteins (Pabo & Sauer, 1984; Brennan & Matthews, 1989). However, we have determined the structure of the E. coli met repressor MetJ. This protein does not contain the helix-turn-helix motif, although there is sequence and structural homology to part of the motif at the C terminus (Rafferty et al., 1989). Model-building suggested that the protein might well bind to DNA by inserting its C-terminal helices into adjacent majo~ grooves on one face of a B-form DNA duplex. However, the structure determination in our laboratory of a complex of MetJ and an operator fragment shows that the opposite face of the molecule, comprising two fl-strands (1 from each monomer), is inserted into the major groove, and side-chains in this region make direct hydrogen-bond contacts to the edges of the base-pairs (Somers et al., unpublished results). This mode of binding seems related to that suggested for arc and mnt repressors on the basis of footprinting and mutagenesis studies (Knight et al., 1989). Such results emphasize the importance of studying a wide range of regulatory proteins, since it is now clear that several such families exist and there are probably more to be discovered. We report here the crystallization of the arginine0o22-283619Ol10o227-02 $03.0ol0
227
© 1990AcademicPressLimited
228
C. W. G. Boys
yielded a homogeneous product (Czaplewski et al., unpublished results). Protein from the chromatography column was precipitated with 28% (w/v) polyethylene glycol 6000 and stored at 4°C. It was subsequently solubilized by "salting-in" to a concentration of 4 mg/ml (determined by its absorbance at 280 nm) in 10 mM-sodium cacodylate/HC1 (pH 7"5) containing 170raM-ammonium sulphate. Crystallization trials by vapour diffusion using 5-]~1 drops on glass or plastic cover-slips were carried out at 17 to 19°C. The wells contained four parts of 100 mM-sodium phosphate buffer (pH 7"6) and one part of 30% (w/v) polyethylene glycol 6000. Rhombohedral crystals up to l'0 mm x0.3 mm x0.3 mm appeared over one week. All solutions contained 25 ~M-L-l-tosylamido-2-phenylethylchloromethyl ketone, 1 mM-phenylmethyl sulphonyl fluoride and 1 mM-dithiothreitol, and were passed through 0"2 #m filters. Crystals have been grown also in the presence of an 80-fold molar excess of L-arginine hydrochloride and 15% (v/v) 2-methyl-2,4-pentane diol in 100mM-phosphate buffer (pH 7"0) and appear as small, triangular rods. The crystals are sensitive to changes in temperature, pH and ionic strength, but are stabilized by the addition of two parts of water to one part of mother liquor. The diffraction pattern extends to a resolution of at least 3"0A (1 A=0.1 nm) with laboratory X-ray sources, and to 2-6A on the Synchrotron Radiation Source at the SERC Daresbury Laboratory. The space group has been determined by precession photography as C2221 with a=229.8/~, b=72.8A and c=137"7A. The cell volume is 2.303 x l06 A a, suggesting that each of the eight asymmetric units per cell probably contains one hexamer. This gives a Vm ratio (Matthews, 1977) of 2-87 ha/Da (solvent content of 55 %), which is in the higher range for protein crystals and may explain their fragility. The presence of a non-crystallographic hexamer implies that phase calculation
and refinement may be possible by using a single heavy-atom derivative. This work has been supported in part by the SERC (GR/E53842 to PGS/SB and GR/E14867 to SEVP) and by the University of Leeds Research Fund. References
Aggarwal, A., Rodgers, D. W., Drottar, M., Ptashne, M. & Harrison, S. C. (1988), Science, 242, 899-907. Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B.W. (1981). Nature (London), 290, 754-758. Brennan, R. G. & Matthews, B. W. (1989). J. Biol. Chem. 264, 1903-1906. Jordan, S. R. & Pabo, C. O. (1988). Science, 242, 893-899. Knight, K. L., Bowie, J. U., Vershon, A. K., Kelley, R. D. & Sauer, R. T. (1989) J. Biol. Chem. 264, 3639-3642. Lim, D., Oppenheim, J. D., Eckhardt, T. & Maas, W. K. (1987). Proe. Nat. Acad. Sci., U.S.A. 84, 6697-6701. Matthews, B. W. (1977). In The Proteins (Neurath, H. & Hill, R.L., eds), 3rd edit., vol. 3, pp. 403-590, Academic Press, New York. Mountain, A. & Baumberg, S. (1980). Mol. Gen. Genet. 178, 691-701. North, A. K. (1989). PhD thesis, Leeds University. North, A. K., Smith, M. C. M. & Baumberg, S. (1989). Gene, 80, 29-38. Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C.L., Joachimiak, A., Marmorstein, R.Q., Luisi, B.F. & Sigler, P. B. (1988). Nature (London), 335, 321-329. Pabo, C.O. & Lewis, M. (1982). Nature (London), 298, 443-447. Pabo, C. O. & Sauer, R. T. (1984). Annu. Rev. Biochem. 53, 293-321. Rafferty, J. B., Somers, W. S., Saint-Girons, I. & Phillips, S. E. V. (1989). Nature (London), 341,705-710 Smith, M. C. M., Czaplewski,m L. G., North, A.K., Baumberg, S. & Stockley, P. G. (1989). Mol. Microbiol. 3, 23-28. Stirling, C. J., Szatmari, G., Stewart, G., Smith, M. C. M. & Sherratt, D.J. (1988). EMBO J. 7, 4389-4398.
Edited by A. Klug
Crystallization of the Arginine-dependent Repressor/Activator AhrC from Bacillus subtilis C. W. G. Boys 1, L. G. Czaplewski 2, S. E. V. Phillips 1, S. Baumberg 2 and P. G. Stockley 2 I Astbury Department of Biophysics 2Department of Genetics University of Leeds, Leeds L S 2 9 J T , U.K. (Received 18 J a n u a r y 1990; accepted 24 J a n u a r y 1990) The arginine-dependent repressor/activator AhrC from Bacillus subtilis has been crystallized in space group C2221, with unit cell dimensions a=229"8 A, b=72-8 A, c= 137"7 A and one aporepressor hexamer per asymmetric unit. Preliminary X-ray photographs show measurable intensities beyond 3"0 A.
dependent r~pressor/activator AhrC from BaciUis subtilis in a form that diffracts to high resolution and may allow a full three-dimensional structure determination. AhrC is the product of the ahrC locus and has been shown to repress the transcription of the arginine biosynthetic enzymes in an arginine-dependent manner, as well as activating the transcription of enzymes involved in arginine catabolism (Mountain & Baumberg, 1980; North et al., 1989). AhrC protein is a hexamer of subunit molecular mass 16"9 kDa (Czaplewski et al., unpublished results). The sequence displays 27 ~ identity with the E. coli homologue ArgR, which has also been shown to be a hexamer (Lim et al., 1987). In addition to regulating arginine biosynthesis in E. coli, ArgR is involved in a site-specific recombination reaction (Stifling et al., 1988). AhrC will complement argR- E. coli strains, although it is not as active as ArgR {Stifling et al., 1988; Smith et al., 1989). No recombination function for the protein in B. subtilis has been discovered, although the gene does lie adjacent to at least one other gene having strong homology with enzymes involved in recombination/repair in E. coli (North, 1989; Van H o y & Hoeh, unpublished results). The AhrC sequence does not contain features characteristic of a helix-turnhelix motif or significant homology to other known repressor proteins, and thus may be representative of a different class of DNA-binding proteins from those studied structurally. The elucidation of its three-dimensional structure should add to our understanding of DNA-protein interactions. Cloned ahrC was overexpressed in E. coli and the protein purified by differential precipitation followed by chromatography on S-Sepharose, which
There are a growing number of known threedimensional structures of bacterial regulatory proteins (Anderson et al., 1981; Pabo & Lewis, 1982), including several complexed with DNA oligonucleotides (Otwinowski et al., 1988; Jordan & Pabo, 1988; Aggarwal et al., 1988). To date, all these structures are of small dimeric molecules isolated from Escherichia coli or its lambda phages and, until very recently, all known structures belonged to the helixturn-helix family of DNA-binding proteins (Pabo & Sauer, 1984; Brennan & Matthews, 1989). However, we have determined the structure of the E. coli met repressor MetJ. This protein does not contain the helix-turn-helix motif, although there is sequence and structural homology to part of the motif at the C terminus (Rafferty et al., 1989). Model-building suggested that the protein might well bind to DNA by inserting its C-terminal helices into adjacent majo~ grooves on one face of a B-form DNA duplex. However, the structure determination in our laboratory of a complex of MetJ and an operator fragment shows that the opposite face of the molecule, comprising two fl-strands (1 from each monomer), is inserted into the major groove, and side-chains in this region make direct hydrogen-bond contacts to the edges of the base-pairs (Somers et al., unpublished results). This mode of binding seems related to that suggested for arc and mnt repressors on the basis of footprinting and mutagenesis studies (Knight et al., 1989). Such results emphasize the importance of studying a wide range of regulatory proteins, since it is now clear that several such families exist and there are probably more to be discovered. We report here the crystallization of the arginine0o22-283619Ol10o227-02 $03.0ol0
227
© 1990AcademicPressLimited
228
C. W. G. Boys
yielded a homogeneous product (Czaplewski et al., unpublished results). Protein from the chromatography column was precipitated with 28% (w/v) polyethylene glycol 6000 and stored at 4°C. It was subsequently solubilized by "salting-in" to a concentration of 4 mg/ml (determined by its absorbance at 280 nm) in 10 mM-sodium cacodylate/HC1 (pH 7"5) containing 170raM-ammonium sulphate. Crystallization trials by vapour diffusion using 5-]~1 drops on glass or plastic cover-slips were carried out at 17 to 19°C. The wells contained four parts of 100 mM-sodium phosphate buffer (pH 7"6) and one part of 30% (w/v) polyethylene glycol 6000. Rhombohedral crystals up to l'0 mm x0.3 mm x0.3 mm appeared over one week. All solutions contained 25 ~M-L-l-tosylamido-2-phenylethylchloromethyl ketone, 1 mM-phenylmethyl sulphonyl fluoride and 1 mM-dithiothreitol, and were passed through 0"2 #m filters. Crystals have been grown also in the presence of an 80-fold molar excess of L-arginine hydrochloride and 15% (v/v) 2-methyl-2,4-pentane diol in 100mM-phosphate buffer (pH 7"0) and appear as small, triangular rods. The crystals are sensitive to changes in temperature, pH and ionic strength, but are stabilized by the addition of two parts of water to one part of mother liquor. The diffraction pattern extends to a resolution of at least 3"0A (1 A=0.1 nm) with laboratory X-ray sources, and to 2-6A on the Synchrotron Radiation Source at the SERC Daresbury Laboratory. The space group has been determined by precession photography as C2221 with a=229.8/~, b=72.8A and c=137"7A. The cell volume is 2.303 x l06 A a, suggesting that each of the eight asymmetric units per cell probably contains one hexamer. This gives a Vm ratio (Matthews, 1977) of 2-87 ha/Da (solvent content of 55 %), which is in the higher range for protein crystals and may explain their fragility. The presence of a non-crystallographic hexamer implies that phase calculation
and refinement may be possible by using a single heavy-atom derivative. This work has been supported in part by the SERC (GR/E53842 to PGS/SB and GR/E14867 to SEVP) and by the University of Leeds Research Fund. References
Aggarwal, A., Rodgers, D. W., Drottar, M., Ptashne, M. & Harrison, S. C. (1988), Science, 242, 899-907. Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B.W. (1981). Nature (London), 290, 754-758. Brennan, R. G. & Matthews, B. W. (1989). J. Biol. Chem. 264, 1903-1906. Jordan, S. R. & Pabo, C. O. (1988). Science, 242, 893-899. Knight, K. L., Bowie, J. U., Vershon, A. K., Kelley, R. D. & Sauer, R. T. (1989) J. Biol. Chem. 264, 3639-3642. Lim, D., Oppenheim, J. D., Eckhardt, T. & Maas, W. K. (1987). Proe. Nat. Acad. Sci., U.S.A. 84, 6697-6701. Matthews, B. W. (1977). In The Proteins (Neurath, H. & Hill, R.L., eds), 3rd edit., vol. 3, pp. 403-590, Academic Press, New York. Mountain, A. & Baumberg, S. (1980). Mol. Gen. Genet. 178, 691-701. North, A. K. (1989). PhD thesis, Leeds University. North, A. K., Smith, M. C. M. & Baumberg, S. (1989). Gene, 80, 29-38. Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C.L., Joachimiak, A., Marmorstein, R.Q., Luisi, B.F. & Sigler, P. B. (1988). Nature (London), 335, 321-329. Pabo, C.O. & Lewis, M. (1982). Nature (London), 298, 443-447. Pabo, C. O. & Sauer, R. T. (1984). Annu. Rev. Biochem. 53, 293-321. Rafferty, J. B., Somers, W. S., Saint-Girons, I. & Phillips, S. E. V. (1989). Nature (London), 341,705-710 Smith, M. C. M., Czaplewski,m L. G., North, A.K., Baumberg, S. & Stockley, P. G. (1989). Mol. Microbiol. 3, 23-28. Stirling, C. J., Szatmari, G., Stewart, G., Smith, M. C. M. & Sherratt, D.J. (1988). EMBO J. 7, 4389-4398.
Edited by A. Klug